Mol Biol Rep (2010) 37:3509–3516 DOI 10.1007/s11033-009-9944-1

The complete mitochondrial genome of the Eupolyphaga sinensis (Blattaria: Polyphagidae) and the phylogenetic relationships within the Dictyoptera

Yan-yan Zhang • Wen-juan Xuan • Jin-liang Zhao • Chao-dong Zhu • Guo-fang Jiang

Received: 28 August 2009 / Accepted: 24 November 2009 / Published online: 10 December 2009 Ó Springer Science+Business Media B.V. 2009

Abstract We present the complete mitochondrial DNA Keywords Complete mitochondrial genome Blattaria sequence of Eupolyphaga sinensis. This closed circular Polyphagidae Eupolyphaga sinensis Dictyoptera molecule is 15553 bp long and consists of 37 genes that Phylogenetic analysis encode for 13 inner membrane proteins, 2 ribosomal RNAs and 22 transfer RNAs. The genome shares the gene order and orientation with previously known Blattaria mito- Introduction chondrial genomes. All tRNAs could be folded into the typical cloverleaf secondary structure, but the tRNASer Complete mitochondrial genome sequences are popular (AGN) appears to be missing the DHU arm. The A ? T- molecular markers used for phylogenetic, phylogeographic rich region is 857 bp long and longer than other cock- and ecological studies in different lineages [1].With roaches. Based on the concatenated amino acid sequences the development of molecular biology techniques, more of all protein coding genes of E. sinensis in conjunction and more mitochondrial genomes have been reported. with those 23 other sequences, we reconstruct Since Wolstenholme and Clary [2] reported the first the phylogenetic tree. Phylogenetic analyses shows that mtDNA sequences, more than 1018 complete metazoa Blataria (including Isoptera) and the Mantodea are sister mitochondrial genomes have been determined. However, groups. Furthermore the relationship of the three basal current knowledge on mtDNAs is very uneven; most clades of winged are different from the three pre- complete mt-genomes have been sequenced for vertebrate vious hypotheses ((Ephemeroptera ? Odonata) ?Neop- taxa available in GenBank (http://www.ncbi.nlm.nih.gov/). tera, Ephemeroptera ? (Odonata ? Neoptera), Odonata ? Insects constitute the most species-rich class among ani- (Ephemeroptera ?Neoptera)). The Ephemeroptera (Para- mals with almost a million of taxa described to date [3], fronurus youi) clusters with the Plecoptera (Pteronarcys but only 114 mitochondrial genomes are from insects princes). (http://www.ncbi.nlm.nih.gov/). Moreover current genomic knowledge of Blattaria is very scanty and the current taxa sampling is extremely poor. In fact, only two species are in Electronic supplementary material The online version of this Blattaria, Periplaneta fuliginosa [4] and Blattella germa- article (doi:10.1007/s11033-009-9944-1) contains supplementary material, which is available to authorized users. nica (unpublished). Eupolyphaga sinensis Walker belongs to the family Po- Y. Zhang W. Xuan J. Zhao G. Jiang (&) lyphagidae and the order Blattaria. This species is widely Jiangsu Key Laboratory for Biodiversity and Biotechnology, distributed in China, but there are few reported in other College of Life Sciences, Nanjing Normal University, Nanjing 210046, China countries. E. sinensis has been used to be a traditional e-mail: [email protected] Chinese medicine. Studies showed that it plays an important role in dealing with thrombi [5, 6]. Despite a lot of studies C. Zhu have been reported, they were limited to the researches on Key Laboratory of Zoological Systematics and Evolution, Chinese Academy of Sciences, Institute of Zoology, morphological, physiological and biological features, few Beijing 100101, China researches on molecular biology have been done yet. 123 3510 Mol Biol Rep (2010) 37:3509–3516

Table 1 Sequencing primers used in the analysis of E. sinensis Primer Upstream primers sequences(50-30) Downstream primers sequences(50-30) Anneal name temperature

1 Ti-J-8:TGCCTGATAAAAAGGRTTAYCKTGATAG C1-N-2329:CTGTAAATATATGATGAGCTC 50°C 2 C1-J-2183:CAACAYTTATTTTGATTYTTTGG TK-N-3785:GTTTAAAGAACCAWTACTTR 50°C 3 C2-J-3640:AAAGCTGATGCAACCCCTGGACGATT N4-N-8700:GGCTTATATTTTAATAATTGCTCATGG 52°C 4 N4-J-8502:GARGGRGGAGCAGCTATATTAS CB-N-11367:ATTACWCCTCCTAATTTRTTAGGAAT 50°C 5 CB-J-11800:CAATGAGTATGAGGAGGATTTGCTGT SR-N-14750:TGTGCCAGCAGTCGCGGTTATACA 50°C 6 SR-J-14588:ATAATAGGTATCTAATCCTAGTTT N2-N-292:TCTAAGCCTGTTCATACACCT 55°C

There are many arguments about the phylogenetic Primer design and PCR amplification relationships among winged insects. The phylogenetic relationship of the Dictyoptera has always been a hot spot. The primers were designed based on universal primers Henning [7] and Kristensen [8] suggest that Blattaria is the [19] and compared with the sequence of B. germanica sister group to Isoptera, forming the group Dictyoptera (unpublished). Six pairs of specific primers were (Mantodea ? (Blattaria ? Isoptera)). But some studies [9– designed to amplify the complete mitochondrial genome 11] are in conflict with their view. They reckon that the in six fragments of approximately 2 kb (1), 1.6 kb (2), phylogenetic relationship of the Dictyoptera should be 5 kb (3), 2.8 kb (4), 2.8 kb (5), 1.5 kb (6), via long PCR Isoptera ? (Blataria ? Mantodea). Besides, the relation- (Table 1). ships among Ephemeroptera, Odonata and Neoptera are Long PCR conditions were the following: an initial ambiguous. As Zhang et al. [12] reported, there are three denaturation for 2 min at 94°C, followed by 15 cycles of main hypotheses as follows: the basal Paleoptera hypoth- denaturation 30 s at 94°C, annealing 30 s at 50–55°C esis ((Ephemeroptera ? Odonata) ? Neoptera), the basal (depending on primer combinations), elongation 60–300 s Ephemeroptera hypothesis (Ephemeroptera ? (Odonat- (depending on putative length of the fragments) at 68°C; a ? Neoptera)) and the basal Odonata hypothesis (Odo- then followed the other PCR program by 15 cycle of nata ? (Ephemeroptera ?Neoptera)). With the application denaturation 30 s at 94°C, annealing 30 s at 50–55°C, of molecular markers, all the three hypotheses were sup- elongation 60–300 s ? 8 s/cycle at 68°C and a final ported by some researchers respectively [12–18]. extension period of 68°C for 10 min. In this study, we were able to sequence and describe the Amplifications were performed on a DNA Engine Ò complete mitochondrial genome of E. sinensis. The newly Peltiter Thermol Cycler (BIO-RAD) in 25 ll reaction determined mtDNA is the first complete sequence for the volume composed of: 13.5 ll of sterilized distilled water, family Polyphagidae and the third for the order Blattaria. 2.5 ll of LA PCR Buffer II (Takara), 2.5 llof25mM On the one hand, the sequences given in the recent study MgCl2,4ll of dNTPs mix, 1 ll of each primer (10 lM), may not only provide useful information to phylogenetic 0.5 ll of DNA template and 0.25 ll (1.25 U) of TaKaRa researches of Blattaria and other insects, but also for LATaq polymerase (Takara). developing mitogenome genetic markers for species iden- tification in the Eupolyphaga species complex. On the Purifying, sequencing and sequence assembling other hand, it may accelerate the researches on molecular biology of E. sinensis. PCR products were tested by electophoresis on an agarose gel. When a single band was observed in the PCR products, long-PCR fragments were purified using the V-gene PCR Materials and methods Clean-up purification Kit. If more than one band present, the appropriately sized PCR product was cut from the gel Samples and DNA extraction and extracted using a Biospin Gel Extraction Kit. Four fragments were sequenced in both directions directly, and Specimens of E. sinensis were sampled from the cockroach large PCR products were sequenced by primer walking isolated from our laboratory. Total genomic DNA was strategy. Two exceptional fragments amplified by N4-J- extracted from the muscle of the fresh specimen’s femora 8502/CB-N-11367 and C2-J-3640/N4-N-8700 were ligated using the standard Proteinase K and phenol/chloroform to the pGEM-T Easy Vector (Promega). Each resulting extraction method. An aliquot of the re-suspended DNA clone was sequenced as well as other four fragments. was used as the template for the subsequent PCR reactions. Sequences were manually checked and assembled using

123 Mol Biol Rep (2010) 37:3509–3516 3511 the software BioEdit and Chromas 2.22 and Seqman (DNASTAR, Steve, 2001).

Data analysis: gene annotation and analysis

Genes encoding proteins, ribosomal RNAs (rRNA) and transfer RNAs (tRNA) were identified according to their amino acid translation or secondary structure features, respectively. Individual gene sequences were compared with the available homologous sequence of Periplaneta fuliginosa in GenBank. The secondary structure of tRNA genes was manually reconstructed using the software DNASIS (Ver.2.5). Protein-coding genes were identified using SEQUIN (Ver.5.35) and compared with other related insects. The nucleotide and amino acid sequences were aligned by ClustalX1.8 [20]. Phylogenetic analysis were performed on the complete amino acid sequences of all the protein Fig. 1 The mitochondrial genome organization of E. sinensis. Genes for proteins and rRNAs are indicated with standard abbreviations, whereas coding genes (PCGs) using a Bayesian approach as those for tRNAs are designated by a single letter for the corresponding implemented in MrBayes 3_0b4 [21]. Following the con- amino acid. Black color is used for the genes (I, M, nad2,W,cox1, clusion of Boore et al. [22] and the results of Carapelli L(UUR), cox2,K,D,atp8, atp6, cox3,G,nad3,A,R,N,S,E,T,nad6, et al. [23], we used the corresponding sequences of cob, S (UCN)) oriented on the J-strand, red for those (Q, C, Y, F, nad5,H, nad4, nad4L,P,nad1,L(CUN),rrnL,V,rrnS) with opposite polarity Pagurus longicarpus and Penaeus mondon of Decapoda as outgroups for rooting. The composition of the j-strand of E. sinensis mtDNA is 31.6% T, 40.4% A, 17.5% C, and 10.5% G, with a total Results and discussion A ? T content of 72.0%.This value is well in the range of other , which show a remarkable variability, from Gene content and genome organization 69.5% to 84.9% A ? T content [27, 28] But it is lower than B. germanica and P. fuliginosa (74.56% and 75.14%). The mtDNA genome of E. sinensis is a circular molecule of 15553 bp. It contains the entire set of 37 genes usually Protein-coding genes and codon usage found in metazoans: 13 PCGs, 22 tRNA genes, and 2 ribosomal genes (Fig. 1; GenBank accession number The mtDNA of E. sinensis contains the full set of PCGs FJ830540). The length in E. sinensis is 557 and 531 bp usually present in animal mtDNA. Canonical initiation longer than that in P. fuliginosam [4] and B. germanica codons (ATA or ATG), encoding the amino acid methio- (unpublished) respectively. The gene order is identical to nine, are used in 8 PCGs (nad2-4, cox2-3, atp6, nad4L, Drosophila melanogaster, in spite of one trnF in D. mel- cob), whereas five other genes start with non-standard anogaster has changed to trnM in E. sinensis. The structure codons (cox1, atp8, nad6, nad1, nad5) (Table 2) as it often of mtDNA is conserved in divergent orders and even happens in animal mtDNAs. It is noteworthy that the some crustaceans [24, 25]. Such an extensive conservation cytochrome oxidase subunit I (cox1) gene often starts with in orientation and gene order in divergent insects has been non-standard codon in other species. It has been exten- inferred to be ancestral among insects [22]. The majority of sively discussed in several arthropod species [29]. Tetra- genes are found on the plus or J-strand, the remainder nucleotides (ATAA, TTAA, and ATTA) and a having opposite polarity and being oriented on the minus or hexanucleotide (ATTTAA) were postulated as the initia- N-strand. The mtDNA genome of E. sinensis also contains tion codon of the cox1 gene, but the initiation codon of the 10 non-coding regions, spanning 1–857 bp. The largest cox1 gene in E. sinensis is the trinucleotide ATT. non-coding region is located at the gene junctions rrnS/ Complete termination codons TAG and TAA were trnI, which is presumed to be homologous to the A ? T- found in three (nad1-3) and six (atp8, atp6, nad5, cox3, rich region of other hexapods by positional homology and nad6, cob) PCGs, respectively. The remaining four genes shared features. This region is considered to regulate and are supposed to end with TA (cox1, cox2) or a single T initiate the replication and transcription of the mitochon- (nad4, nad4L). Not surprisingly, their function would be drial genome [26]. recovered into a complete TAA stop codon by a post- 123 3512 Mol Biol Rep (2010) 37:3509–3516

Table 2 Annotation, nucleotide composition and other features of the mitochondrial genome of E. sinensis Gene Strand Sites Seq position %T %C %A %G %A ? T Start codon Stop codon trnI J 69 1–69 34.8 11.6 33.3 20.3 68.1 trnQ N 69 67–135 44.9 5.8 31.9 17.4 76.8 trnM J 68 144–211 25 22.1 38.2 14.7 63.2 nd2 J 1020 212–1231 34.4 18.7 36.2 10.7 70.6 ATG TAG trnW J 67 1236–1302 31.3 14.9 44.8 9 76.1 trnC N 68 1295–1362 33.8 10.3 38.2 17.6 72 trnY N 66 1363–1428 31.8 9.1 33.3 25.8 65.1 cox1 J 1544 1430–2973 35.9 17.2 31 15.9 66.9 ATT TAN trnL(UUR) J 70 2974–3043 34.3 12.9 35.7 17.1 70 cox2 J 680 3049–3728 34.1 17.5 35.6 12.8 69.7 ATG TAN trnK J 70 3729–3798 30 17.1 38.6 14.3 68.6 trnD J 68 3800–3867 36.8 10.3 41.2 11.8 78 atp8 J 156 3868–4023 34.6 16.7 44.2 4.5 78.8 ATT TAA atp6 J 681 4017–4697 37.7 16.2 36 10.1 73.7 ATG TAA cox3 J 789 4697–5485 34.3 18.4 32.8 14.4 67.1 ATG TAA trnG J 63 5485–5547 33.3 9.5 49.2 7.9 82.5 nd3 J 354 5545–5898 36.4 18.4 33.9 11.3 70.3 ATA TAG trnA J 63 5897–5959 34.9 9.5 41.3 14.3 76.2 trnR J 63 5959–6021 39.7 12.7 38.1 9.5 77.8 trnN J 66 6020–6085 33.3 10.6 45.5 10.6 78.8 trnS(AGN) J 68 6085–6152 35.3 13.2 30.9 20.6 66.2 trnE J 65 6152–6216 40 10.8 44.6 4.6 84.6 trnF N 70 6215–6284 35.7 4.3 41.4 18.6 77.1 nd5 N 1731 6279–8009 46.7 9.3 26.3 17.7 73 GTG TAA trnH N 65 8010–8074 47.7 4.6 29.2 18.5 76.9 nd4 N 1345 8063–9407 48.5 9 24.8 17.8 73.3 ATG TNN nd4L N 280 9406–9685 51.1 6.8 27.5 14.6 78.6 ATG TNN trnT J 63 9688–9750 38.1 7.9 42.9 11.1 81 trnP N 64 9751–9814 45.3 4.7 32.8 17.2 78.1 nd6 J 495 9817–10311 31.9 16.2 42.2 9.7 74.1 ATT TAA cob J 1134 10311–11444 35.7 18.7 33.2 12.4 68.9 ATG TAA trnS(UCN) J 67 11442–11508 35.8 7.5 43.3 13.4 79.1 nd1 N 921 11524–12444 49.5 9.8 20.7 20 70.2 ATT TAG trnL(CUN) N 69 12465–12533 34.8 10.1 36.2 18.8 71 rrnL N 1293 12534–13826 44.9 6.9 29.8 18.4 74.7 trnV N 69 13827–13895 39.1 11.6 31.9 17.4 71 rrnS N 801 13896–14696 43.4 8.9 29.3 18.4 72.7 A ? T-rich 857 14697–15553 37.6 14.9 40.3 7.2 77.9 transcriptional polyadenylation [30]. Three (cox1, cox2, implicit assumption that synonymous codons are equally nad4) are exactly adjacent to tRNAs, and one (nad4L)is used and most values differ from 1 (frequency at exactly adjacent to the beginning of another gene (nad4). equilibrium). Thus, it is highly probable that during mRNA processing the U is exposed at the end of an mRNA molecule and Transfer RNAs polyadenylated, forming the complete UAA [31]. Relative synonymous codon usage [32] values were All the 22 typical tRNA genes were identified in the calculated for the PCGs (Table S1, Supplementary Mate- mtDNA of E. sinensis according to their secondary struc- rial). Obviously, codon usage is out of harmony with the tures and primary sequences of the corresponding

123 Mol Biol Rep (2010) 37:3509–3516 3513 anticodon (Fig. S1, Supplementary Material). The 22 tRNA Friesea grisea (NC_010535)[36], Cryptopygus antarcticus genes were interspersed in the genome and range from 63 (NC_010533) [1], Orchesella villosa (NC_010534) [36], to 70 bp in length. Apart from trnS (AGN), all of them Tamolanica tamolana (NC_007702) [39], Sclerophasma showed typical clover-leaf secondary structures and their paresisense(NC_007701) [39], Periplaneta fuliginosa anticodons are similar to those found in other metazoan (NC_006076) [4], Blattella germanica (EU854321; unpub- . The only peculiar feature is the lack of the DHU lished), Thermobia domestica (NC_006080) [40], Atelura arm in trnS (AGN). This feature is shared with some other formicaria (NC_011197) [41], Locusta migratoria (NC_011119; animals, but is not a general feature of Blattaria mtDNA as unpublished), Calliptamus italicus (NC_011305) [42], proved by B. germanica which has all tRNAs with a Parafronurus youi (NC_011359) [12], Campodea lubbocki complete clover-leaf structure (unpublished). Though it (NC_008234) [43], Japyx solifugus (NC_007214) [44], can’t show the typical clover-leaf secondary structure, it Penaeus monodon (NC_002184) [45] and Pagurus longi- can form the correct tertiary structure. Moreover, overlaps carpus (NC_003058) [46]. between trnW and trnC genes were 8 bp, as reported by Every set PCG sequences was concatenated gene-by- Lessinger et al. [33] which makes the genes producing gene and produced 24 sets sequences of PCGs, then aligned separate transcripts with their opposite directions. them by ClustalX1.8 [20]. After removing the unreliable alignment sites and gaps manually inspected, the final alignment resulted in 2974 amino acid sites, 726 were Ribosomal RNAs constant, 2248 were variable, and 1581 were parsimony informative. As reported for other animals, two genes for ribosomal What’s the phylogenetic relationships among Dictyop- RNAs were fond in E. sinensis, one for the small (srRNA) tera: Mantodea ? (Blattaria ? Isoptera) or Isoptera ? and one for the large ribosomal subunit (lrRNA). The (Blataria ? Mantodea)? The argument has been lasting for srRNA was located between trnV and A ? T-rich region, many years. Some studies confirmed the first postulate [7, 8], and the lrRNA was located between trnV and trnL. but were in conflict with others [9–11]. However, DeSalle et al. [47] reckoned that Mantodea and Isoptera were the A ? T-rich region (control region) sister groups. But in this study, a peculiar finding of this analysis is that E. sinensis clusters with Isoptera insects The A ? T-rich region in E. sinensis mtDNA, comprising (Reticulitermes flavipes and R. virginicus), then they cluster the region between srRNA and trnI was 857 bp long. This with another Blattaria insect (P. fuliginosa). In another word, length was well in the range of other arthropods, which all the Blattaria insects do not cluster together. As reported shows a remarkable variability; from 263nt for Rhipi- by Inward et al.[48], molecular phylogenetic analysis cephalus to 4,601nt for D. melanogaster [34]. It is 604 bp showed that termites are social , no longer and 649 bp longer than P. fuliginosa and B. germanica. meriting being classified as separate order (Isoptera) from Previous analysis of the Drosophila A ? T-rich region the cockroaches (). Instead, they proposed that they identified the signals responsible for the origin of replica- should be treated as a family i.e.Termitidae within Blattaria. tion of the major and minor strands (OJ and ON, respec- Obviously our study supplied the molecular evidences for tively) and confirmed that, at least in this genus, the the postulate (Fig. 2). Since the Isoptera group died [48], we mtDNA replicates with a strand-asynchronous, asymmetric proposed that the Blataria (including Isoptera) and the mechanism. Mantodea are sister groups. Our phylogenetic tree reflected the most widely accepted Phylogenetic analysis interpretation, based on morphological and molecular data, the basal splitting of the Insecta separates the Phylogenetic analysis were performed on the concatenated from the Dicondylia (Zygentoma ? Pterygota) [8, 13, 16, amino acid sequences of all PCGs of E. sinensis in con- 17, 36]. Within Archaeognatha and Zygentoma, relation- junction with those 23 other arthropod sequences whose ships are stable across different analysis and congruent with complete mtDNA sequences were available in GenBank, the accepted . But the relationships among using a Bayesian approach implemented in MrBayes 3_0b4 Ephemeroptera, Odonata and Neoptera are ambiguous. For [21]. The sequences were as follows: Reticulitermes vir- them, three hypotheses were found as before. With the ginicus (NC_009500) [23], R.flavipes (NC_009498) [23], application of molecular markers, all the three hypotheses Pteronarcys princes (NC_006133) [35], Orthetrum trian- were supported by some researches respectively [14–16, 18]. gulare melania (AB126005) [4], Trigoniophthalmus al- As Ogden and Whiting [15] reported, their result based on ternatus (NC_010532) [36], Nesomachilis australica 18S rDNA, 28S rDNA and the Histone 3 protein-coding gene (NC_006895) [37], Petrobius brevistylis (NC_007688) [38], (H3) supported the basal Ephemeroptera hypothesis. But 123 3514 Mol Biol Rep (2010) 37:3509–3516

Fig. 2 Phylogenetic 1.00 Reticulitermes virginicus relationships of insects inferred 1.00 Reticulitermes flavipes Isoptera from amino acid sequences of 13 mitochondrial protein-coding 0.97 Eupolyphag sinensis genes of 22 Hexapoda by 1.00 Bayesian analysis. Tree was Periplaneta fuliginosa Blattaria 1.00 rooted by branchipod crustacean Blattella germanica Pagurus longicarpus and 1.00 Penaeus mondon. Numbers at Tamolanica tamolana Mantodea nodes indicate posterior 1.00 Sclerophasma paresisense probabilities (9100). Vertical Mantophasmatodea lines indicate monophyletic 1.00 Calliptamus italicus orders 1.00 Locusta migratoria Orthoptera

1.00 Parafronurus youi Ephemeroptera 1.00 Pteronarcys princes Plecoptera 1.00 Orthetrum triangulare melania Odonata Atelura formicaria 1.00 1.00 Thermobia domestica Zygentoma

Petrobius brevistylis 1.00 0.53 1.00 Trigoniophthalmus alternatus Archaeognatha Nesomachilis australica

Japyx solifugus 1.00 0.53 Campodea lubbocki Campodeoidea Cryptopygus antarcticus 1.00 1.00 Orchesella villosa Collembola Friesea grisea

Pagurus longicarpus

Penaeus monodon other 18S/28S analysis supported the basal Palaeoptera solifugus ? Campodea lubbocki and Collembola ? (Campo- hypothesis [14]. Also the basal Odonata hypothesis had been deoidea ? Insecta) were poorly supported (53%, both) in supported by different molecular markers: 18S rRNA [16, analysis, suggesting that further researches on the rela- 18, 49] and combined complete 18S rRNA and 28S rRNA tionships of Campodeoidea and Collembola should be [17]. Furthermore, based on amino acid data, Carapelli carried on (Fig. 2). et al.[36] proposed that the odonatan is the basal lineage of the Pterygota. Unfortunately, the absence of the Epheme- roptera from their analysis prevents them to draw conclu- Conclusion sions of the relationships. Zhang et al. [12] first sequenced the complement complete mitochondrial genome of Parafronurus The mitochondrial genome of E. sinensis is the first youi. Their phylogenetic tree based on 12 mitochondrial sequenced mtDNA for a representative of the Polyphagidae PCGs supported the basal Ephemeroptera hypotheses and the third for the Order Blattaria. The newly determined (Ephemeroptera ? (Odonata ? Neoptera)). Surprisingly, genome shares the gene order and orientation with previ- our study is different from the three hypotheses. The ously known Blattaria genomes. It is rich in A ? T Ephemeroptera (Parafronurus youi) clusters with the Ple- throughout the entire mitochondrial genomes. The phylo- coptera (Pteronarcys princes) (Fig. 2). Hence, we think the genetic analysis shows that Blataria (including Isoptera) relationships among Ephemeroptera, Odonata and Neoptera and Mantodea are sister groups. Furthermore the relation- should be studied further. ships of the three basal clades of winged insects are A monophyletic Collembola was recovered. Low or no different from the three previous hypotheses ((Ephemerop- support was found for the deeper nodes, including the tera ? Odonata) ? Neoptera, Ephemeroptera ? (Odonat- monophyly of the Collembola. Two nodes, Japyx a ? Neoptera), Odonata ? (Ephemeroptera ? Neoptera)).

123 Mol Biol Rep (2010) 37:3509–3516 3515

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